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Summary: Imagine if we could simultaneously predict spatial protein expression in tissues from their routine Hematoxylin and Eosin (H&E) stained images, and create tissue images given protein expression profiles thus enabling virtual simulations of how protein expression alterations impact histology in complex diseases like cancer. Such an approach could lead to more informed diagnostic and therapeutic decisions for precision medicine at lower costs and shorter turnaround times, more detailed insights into underlying disease pathology as well as improvement in predictive and generative performance. In this study, we investigate the intricate correlation between protein expressions obtained from Hyperion mass cytometry and histopathological microstructures in conventional H&E stained glioblastoma (GBM) samples, unveiling morphological patterns and cellular-level spatial alterations associated with protein expression changes. To model these complex relationships, we propose a novel generative-predictive framework called Ouroboros for producing H&E images from protein expressions and simultaneously predicting protein expressions from H&E images. Our comprehensive sample-independent validation over 9920 tissue spots from 4 GBM samples encompassing visual image analysis, quantitative analysis, subspace alignment and perturbation experiments shows that the proposed generative-predictive approach offers significant improvements in predicting protein expression from images in comparison to baseline methods as well as accurate generation of virtual GBM sample images. This proof of concept study can contribute to advancing our understanding of histological responses to protein expression perturbations and lays the foundations for further developments in this area., Availability and Implementation: Implementation and associated data for the proposed approach are available at the URL: https://github.com/Srijay/Ouroboros., (© The Author(s) 2024. Published by Oxford University Press.)

Cationic polysaccharides have been extensively studied for drug delivery via the bloodstream, yet few have progressed to clinical use. Endothelial cells lining the blood vessel wall are coated in an anionic extracellular matrix called the glycocalyx. However, we do not fully comprehend the charged polysaccharide interactions with the glycocalyx. We reveal that the cationic polysaccharide poly(acetyl, arginyl) glucosamine (PAAG) exhibits the highest association with the endothelial glycocalyx, followed by dextran (neutral) and hyaluronan (anionic). Furthermore, we demonstrate that PAAG binds heparan sulfate (HS) within the glycocalyx, leading to intracellular accumulation. Using an in vitro glycocalyx model, we demonstrate a charge-based extent of association of polysaccharides with HS. Mechanistically, we observe that PAAG binding to HS occurs via a condensation reaction and functionally protects HS from degradation. Together, this study reveals the interplay between polysaccharide charge properties and interactions with the endothelial cell glycocalyx toward improved delivery system design and application.

Melioidosis is an emerging tropical infection caused by inhalation, inoculation, or ingestion of the flagellated, facultatively intracellular pathogen Burkholderia pseudomallei . The melioidosis case fatality rate is often high, and pneumonia, the most common presentation, doubles the risk of death. The alveolar macrophage is a sentinel pulmonary host defense cell, but the human alveolar macrophage in B. pseudomallei infection has never been studied. The objective of this study was to investigate the host-pathogen interaction of B. pseudomallei infection with the human alveolar macrophage and to determine the role of flagellin in modulating inflammasome-mediated pathways. We found that B. pseudomallei infects primary human alveolar macrophages but is gradually restricted in the setting of concurrent cell death. Electron microscopy revealed cytosolic bacteria undergoing division, indicating that B. pseudomallei likely escapes the alveolar macrophage phagosome and may replicate in the cytosol, where it triggers immune responses. In paired human blood monocytes, uptake and intracellular restriction of B. pseudomallei are similar to those observed in alveolar macrophages, but cell death is reduced. The alveolar macrophage cytokine response to B. pseudomallei is characterized by marked interleukin (IL)-18 secretion compared to monocytes. Both cytotoxicity and IL-18 secretion in alveolar macrophages are partially flagellin dependent. However, the proportion of IL-18 release that is driven by flagellin is greater in alveolar macrophages than in monocytes. These findings suggest differential flagellin-mediated inflammasome pathway activation in the human alveolar macrophage response to B. pseudomallei infection and expand our understanding of intracellular pathogen recognition by this unique innate immune lung cell., Competing Interests: The authors declare no conflict of interest.

Immune cell dysfunction within the tumor microenvironment (TME) undermines the control of cancer progression. Established tumors contain phenotypically distinct, tumor-specific natural killer (NK) cells; however, the temporal dynamics, mechanistic underpinning and functional significance of the NK cell compartment remains incompletely understood. Here, we use photo-labeling, combined with longitudinal transcriptomic and cellular analyses, to interrogate the fate of intratumoral NK cells. We reveal that NK cells rapidly lose effector functions and adopt a distinct phenotypic state with features associated with tissue residency. NK cell depletion from established tumors did not alter tumor growth, indicating that intratumoral NK cells cease to actively contribute to anti-tumor responses. IL-15 administration prevented loss of function and improved tumor control, generating intratumoral NK cells with both tissue-residency characteristics and enhanced effector function. Collectively, our data reveals the fate of NK cells after recruitment into tumors and provides insight into how their function may be revived., (© 2024. The Author(s).)

Background: Mucus stasis, a hallmark of muco-obstructive disease, results from impaired mucociliary transport and leads to lung function decline and chronic infection. Although therapeutics that target mucus stasis in the airway, such as hypertonic saline or rhDNAse, show some therapeutic benefit, they do not address the underlying electrostatic defect apparent in mucins in CF and related conditions. We have previously shown poly (acetyl, arginyl) glucosamine (PAAG, developed as SNSP113), a soluble, cationic polymer, significantly improves mucociliary transport in a rat model of CF by normalizing the charge defects of CF mucin. Here, we report efficacy in the CFTR-sufficient, ENaC hyperactive, Scnn1b-Tg mouse model that develops airway muco-obstruction due to sodium hyperabsorption and airway dehydration., Methods: Scnn1b-Tg mice were treated with either 250 µg/mL SNSP113 or vehicle control (1.38% glycerol in PBS) via nebulization once daily for 7 days and then euthanized for analysis. Micro-Optical Coherence Tomography-based evaluation of excised mouse trachea was used to determine the effect on the functional microanatomy. Tissue analysis was performed by routine histopathology., Results: Nebulized treatment of SNSP113 significantly improved mucociliary transport in the airways of Scnn1b-Tg mice, without altering the airway surface or periciliary liquid layer. In addition, SNSP113 significantly reversed epithelial hypertrophy and goblet cell metaplasia. Finally, SNSP113 significantly ameliorated eosinophilic crystalline pneumonia and lung consolidation in addition to inflammatory macrophage influx in this model., Conclusion: Overall, this study extends the efficacy of SNSP113 as a potential therapeutic to alleviate mucus stasis in muco-obstructive diseases in CF and potentially in related conditions., Competing Interests: Declaration of Competing Interest Steven M. Rowe received grant funding and consulting fees from Synspira and Synedgen that ended in 2022. Shenda M. Baker provided SNSP113 (PAAG) used in study. Elex S. Harris, Lea Novak, Courtney M. Fernandez-Petty, and Susan E. Birket have no conflicts of interest to report., (Copyright © 2023. Published by Elsevier B.V.)

During embryonic development, all blood progenitors are initially generated from endothelial cells that acquire a hemogenic potential. Blood progenitors emerge through an endothelial-to-hematopoietic transition regulated by the transcription factor RUNX1. To date, we still know very little about the molecular characteristics of hemogenic endothelium and the molecular changes underlying the transition from endothelium to hematopoiesis. Here, we analyzed at the single cell level a human embryonic stem cell-derived endothelial population containing hemogenic potential. RUNX1-expressing endothelial cells, which harbor enriched hemogenic potential, show very little molecular differences to their endothelial counterpart suggesting priming toward hemogenic potential rather than commitment. Additionally, we identify CD82 as a marker of the endothelium-to-hematopoietic transition. CD82 expression is rapidly upregulated in newly specified blood progenitors then rapidly downregulated as further differentiation occurs. Together our data suggest that endothelial cells are first primed toward hematopoietic fate, and then rapidly undergo the transition from endothelium to blood., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

CD146, also known as melanoma cell adhesion molecule (MCAM), is expressed in numerous cancers and has been implicated in the regulation of metastasis. We show that CD146 negatively regulates transendothelial migration (TEM) in breast cancer. This inhibitory activity is reflected by a reduction in MCAM gene expression and increased promoter methylation in tumour tissue compared to normal breast tissue. However, increased CD146/MCAM expression is associated with poor prognosis in breast cancer, a characteristic that is difficult to reconcile with inhibition of TEM by CD146 and its epigenetic silencing. Single cell transcriptome data revealed MCAM expression in multiple cell types, including the malignant cells, tumour vasculature and normal epithelium. MCAM expressing malignant cells were in the minority and expression was associated with epithelial to mesenchymal transition (EMT). Furthermore, gene expression signatures defining invasiveness and a stem cell-like phenotype were most strongly associated with mesenchymal-like tumour cells with low levels of MCAM mRNA, likely to represent a hybrid epithelial/mesenchymal (E/M) state. Our results show that high levels of MCAM gene expression are associated with poor prognosis in breast cancer because they reflect tumour vascularisation and high levels of EMT. We suggest that high levels of mesenchymal-like malignant cells reflect large populations of hybrid E/M cells and that low CD146 expression on these hybrid cells is permissive for TEM, aiding metastasis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mannion, Odell, Baker, Matthews, Jones and Cook.)

Cell membranes are key interfaces where materials engineering meets biology. Traditionally regarded as just the location of receptors regulating the uptake of molecules, we now know that all mammalian cell membranes are 'sugar coated'. These sugars, or glycans, form a matrix bound at the cell membrane via proteins and lipids, referred to as the glycocalyx, which modulate access to cell membrane receptors crucial for interactions with drug delivery systems (DDS). Focusing on the key blood-tissue barrier faced by most DDS to enable transport from the place of administration to target sites via the circulation, we critically assess the design of carriers for interactions at the endothelial cell surface. We also discuss the current challenges for this area and provide opportunities for future research efforts to more fully engineer DDS for controlled, efficient, and targeted interactions with the endothelium for therapeutic application., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In vitro generation and expansion of hematopoietic stem cells (HSCs) holds great promise for the treatment of any ailment that relies on bone marrow or blood transplantation. To achieve this, it is essential to resolve the molecular and cellular pathways that govern HSC formation in the embryo. HSCs first emerge in the aorta-gonad-mesonephros (AGM) region, where a rare subset of endothelial cells, hemogenic endothelium (HE), undergoes an endothelial-to-hematopoietic transition (EHT). Here, we present full-length single-cell RNA sequencing (scRNA-seq) of the EHT process with a focus on HE and dorsal aorta niche cells. By using Runx1b and Gfi1/1b transgenic reporter mouse models to isolate HE, we uncovered that the pre-HE to HE continuum is specifically marked by angiotensin-I converting enzyme (ACE) expression. We established that HE cells begin to enter the cell cycle near the time of EHT initiation when their morphology still resembles endothelial cells. We further demonstrated that RUNX1 AGM niche cells consist of vascular smooth muscle cells and PDGFRa+ mesenchymal cells and can functionally support hematopoiesis. Overall, our study provides new insights into HE differentiation toward HSC and the role of AGM RUNX1+ niche cells in this process. Our expansive scRNA-seq datasets represents a powerful resource to investigate these processes further., (© 2022 by The American Society of Hematology.)

Pseudomonas aeruginosa is a common opportunistic pathogen that can cause chronic infections in multiple disease states, including respiratory infections in patients with cystic fibrosis (CF) and non-CF bronchiectasis. Like many opportunists, P. aeruginosa forms multicellular biofilm communities that are widely thought to be an important determinant of bacterial persistence and resistance to antimicrobials and host immune effectors during chronic/recurrent infections. Poly (acetyl, arginyl) glucosamine (PAAG) is a glycopolymer that has antimicrobial activity against a broad range of bacterial species, and also has mucolytic activity, which can normalize the rheological properties of cystic fibrosis mucus. In this study, we sought to evaluate the effect of PAAG on P. aeruginosa bacteria within biofilms in vitro , and in the context of experimental pulmonary infection in a rodent infection model. PAAG treatment caused significant bactericidal activity against P. aeruginosa biofilms, and a reduction in the total biomass of preformed P. aeruginosa biofilms on abiotic surfaces, as well as on the surface of immortalized cystic fibrosis human bronchial epithelial cells. Studies of membrane integrity indicated that PAAG causes changes to P. aeruginosa cell morphology and dysregulates membrane polarity. PAAG treatment reduced infection and consequent tissue inflammation in experimental P. aeruginosa rat infections. Based on these findings we conclude that PAAG represents a novel means to combat P. aeruginosa infection, and may warrant further evaluation as a therapeutic.

High levels of histone acetylation are associated with the regulatory elements of active genes, suggesting a link between acetylation and gene activation. We revisited this model, in the context of EGF-inducible gene expression and found that rather than a simple unifying model, there are two broad classes of genes; one in which high lysine acetylation activity is required for efficient gene activation, and a second group where the opposite occurs and high acetylation activity is inhibitory. We examined the latter class in more detail using EGR2 as a model gene and found that lysine acetylation levels are critical for several activation parameters, including the timing of expression onset, and overall amplitudes of the transcriptional response. In contrast, DUSP1 responds in the canonical manner and its transcriptional activity is promoted by acetylation. Single cell approaches demonstrate heterogenous activation kinetics of a given gene in response to EGF stimulation. Acetylation levels modify these heterogenous patterns and influence both allele activation frequencies and overall expression profile parameters. Our data therefore point to a complex interplay between acetylation equilibria and target gene induction where acetylation level thresholds are an important determinant of transcriptional induction dynamics that are sensed in a gene-specific manner., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background and Aims: Palliative care is a critical component of the response of a healthcare system to a pandemic. We present risk factors associated with mortality and highlight an operational palliative care consult service in facilitating early identification of risk factors to guide goal-concordant care and rational utilization of finite healthcare resources during a pandemic., Methods: In this case series of 100 consecutive patients hospitalized with COVID-19, we analyzed clinical data, treatment including palliative care, and outcomes in patients with SARS-CoV-2 infection admitted to three hospitals in Seattle, Washington. We compared data between patients who were discharged and non-survivors., Results: Age (OR 4.67 [1.43, 15.32] ages 65-79; OR 3.96 [1.05, 14.89] ages 80-97), dementia (OR 5.62 [1.60, 19.74]), and transfer from a congregate living facility (OR 5.40 [2.07, 14.07]), as well hypoxemia and tachypnea (OR 7.00 [2.91, 22.41]; OR 2.78 [1.11, 6.97]) were associated with mortality. Forty-one (41%) patients required intensive care and 22 (22%) invasive mechanical ventilation. Forty-six (46%) patients were seen by the palliative care service, resulting in a change of resuscitation status in 54% of admitted patients. Fifty-eight (58%) patients recovered and were discharged, 34 (34%) died, and eight (8%) remained hospitalized, of which seven ultimately survived and one died., Conclusions: Older age, dementia, and congregate living were associated with mortality. Early discussions of goals of care facilitated by an operational palliative care consult service can effectively guide goal-concordant care in patients at high risk for mortality during a pandemic. Development of a functional palliative care consult service is an important component of pandemic planning., Competing Interests: The authors declare no conflicts of interest., (© 2021 The Authors. Health Science Reports published by Wiley Periodicals LLC.)

Innate lymphoid cells (ILCs) are guardians of mucosal immunity, yet the transcriptional networks that support their function remain poorly understood. We used inducible combinatorial deletion of key transcription factors (TFs) required for ILC development (RORγt, RORα and T-bet) to determine their necessity in maintaining ILC3 identity and function. Both RORγt and RORα were required to preserve optimum effector functions; however, RORα was sufficient to support robust interleukin-22 production among the lymphoid tissue inducer (LTi)-like ILC3 subset, but not natural cytotoxicity receptor (NCR) + ILC3s. Lymphoid tissue inducer-like ILC3s persisted with only selective loss of phenotype and effector functions even after the loss of both TFs. In contrast, continued RORγt expression was essential to restrain transcriptional networks associated with type 1 immunity within NCR + ILC3s, which coexpress T-bet. Full differentiation to an ILC1-like population required the additional loss of RORα. Together, these data demonstrate how TF networks integrate within mature ILCs after development to sustain effector functions, imprint phenotype and restrict alternative differentiation programs., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Xenopus tadpoles have the ability to regenerate their tails upon amputation. Although some of the molecular and cellular mechanisms that globally regulate tail regeneration have been characterised, tissue-specific response to injury remains poorly understood. Using a combination of bulk and single-cell RNA sequencing on isolated spinal cords before and after amputation, we identify a number of genes specifically expressed in the spinal cord during regeneration. We show that Foxm1, a transcription factor known to promote proliferation, is essential for spinal cord regeneration. Surprisingly, Foxm1 does not control the cell cycle length of neural progenitors but regulates their fate after division. In foxm1 -/- tadpoles, we observe a reduction in the number of neurons in the regenerating spinal cord, suggesting that neuronal differentiation is necessary for the regenerative process. Altogether, our data uncover a spinal cord-specific response to injury and reveal a new role for neuronal differentiation during regeneration., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Background: Biofilms are microbial communities surrounded by a self-produced extracellular matrix which protects them from environmental stress. Bacteria within biofilms are 10- to 1000-fold more resistant to antibiotics, making it challenging but imperative to develop new therapeutics that can disperse biofilms and eradicate infection. Gram-negative bacteria produce outer membrane vesicles (OMV) that play critical roles in communication, genetic exchange, cargo delivery, and pathogenesis. We have previously shown that OMVs derived from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi., Results: Here, we examine the antibiofilm activity of Burkholderia thailandensis OMVs against the oral biofilm-forming pathogen Streptococcus mutans. We demonstrate that OMV treatment reduces biofilm biomass, biofilm integrity, and bacterial cell viability. Both heat-labile and heat-stable components, including 4-hydroxy-3-methyl-2-(2-non-enyl)-quinoline and long-chain rhamnolipid, contribute to the antibiofilm activity of OMVs. When OMVs are co-administered with gentamicin, the efficacy of the antibiotic against S. mutans biofilms is enhanced., Conclusion: These studies indicate that bacterial-derived OMVs are highly effective biological nanoparticles that can inhibit and potentially eradicate biofilms., (© 2021. The Author(s).)

Pericardial constriction can be present without pericardial calcium and often without pericardial thickening. This epicardial coronary artery motion abnormality due to entrapment in a thickened, fibrotic pericardium, is characteristic of constrictive pericarditis, and differentiates this entity from other close differential diagnoses, such as restrictive and dilated cardiomyopathy.

About 90% of grapefruit in Florida are affected by Huanglongbing (HLB). HLB negatively affects the organoleptic properties of grapefruit juice because affected trees overproduce bitter secondary-metabolites, mostly naringin. The objective of this research was to remove naringin from HLB-affected grapefruit juice using microporous-adsorbents and to investigate how debittering affected narirutin, limonoids, bergamottin, and consumer acceptability. The adsorption kinetics of naringin on seven adsorbent resins obeyed pseudo-second order. PAD550 and PAD600 showed better static adsorption/desorption. Adsorption-isotherms on these resins were better fitted on Temkin-Pyzhev-model. On a fixed-bed-column packed with PAD550 resin, a slower loading rate increased its breakthrough volume before naringin in effluent reached its taste-threshold. In addition to naringin being reduced to below its taste-threshold, debittering significantly decreased the content of limonin, nomilin, and bergamottin. A consumer taste panel rated debittered and half-debittered juices higher for overall acceptability than the untreated. The half-debittered juice was ranked the most preferred while untreated was the least preferred., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Tuna (Thunnus albacares) and mahi-mahi (Coryphaena hippurus) are two major fish species responsible for scombroid poisoning in the United States. The purpose of this research was to develop a low-cost and easily operated colorimetric strip method for the rapid determination of spoilage degree via amine response in mahi-mahi and tuna. The color strip method was developed by investigating different types of dyes, filter papers, sample volume, water bath temperature, and other parameters. Ultimately rose bengal and bromophenol blue (BPB) dyes were chosen. These two dyes produced standard curves with good linearity (0-50 mg/L for the total biogenic amines) and uniformity of color change. The r 2 values for the standard curves of the rose Bengal and BPB were 0.9535 and 0.8883, respectively. Significant positive Pearson correlations coefficients (r) between the volatile biogenic amine levels detected by these two colorimetric strip methods with increasing spoilage grade of mahi-mahi (rose bengal: r = 0.8907, p < 0.0001; BPB: r = 0.8711, p < 0.0001) and tuna (rose bengal: r = 0.8351, p < 0.0001; BPB: r = 0.7362, p = 0.0001) were observed. For mahi-mahi, the volatile amines detected by the colorimetric strips correlated positively with increasing levels of eight biogenic amines, free alanine, four aldehydes, isoamyl alcohol, two ketones, and dimethyl disulfide. For tuna, the results determined by colorimetric strips positively correlated with three biogenic amines, three free amino acids, four aldehydes, and ethanol. The two validated colorimetric strips could rapidly monitor the spoilage degree of mahi-mahi and tuna at low-cost. PRACTICAL APPLICATION: Rose bengal strips and BPB strips were developed as a rapid, objective, analytical method that can serve as an alternative to sensory grading methods. These two nonspecific colorimetric strip methods provided good linear response and uniformity of color change. Volatile amine levels in fish determined by these colorimetric strip methods were statistically significant and positively correlated with the spoilage grade of fish., (© 2021 Institute of Food Technologists®.)

Burkholderia pseudomallei is a Gram-negative, facultative intracellular bacillus that causes the disease melioidosis. B. pseudomallei expresses a number of proteins that contribute to its intracellular survival in the mammalian host. We previously demonstrated that immunization with OMVs derived from B. pseudomallei grown in nutrient-rich media protects mice against lethal disease. Here, we evaluated if OMVs derived from B. pseudomallei grown under macrophage-mimicking growth conditions could be enriched with intracellular-stage proteins in order to improve the vaccine. We show that OMVs produced in this manner (M9 OMVs) contain proteins associated with intracellular survival yet are non-toxic to living cells. Immunization of mice provides significant protection against pulmonary infection similar to that achieved with a live attenuated vaccine and is associated with increased IgG, CD4 + , and CD8 + T cells. OMVs possess inherent adjuvanticity and drive DC activation and maturation. These results indicate that M9 OMVs constitute a new promising vaccine against melioidosis.

Gene expression programs determine cell fate in embryonic development and their dysregulation results in disease. Transcription factors (TFs) control gene expression by binding to enhancers, but how TFs select and activate their target enhancers is still unclear. HOX TFs share conserved homeodomains with highly similar sequence recognition properties, yet they impart the identity of different animal body parts. To understand how HOX TFs control their specific transcriptional programs in vivo, we compared HOXA2 and HOXA3 binding profiles in the mouse embryo. HOXA2 and HOXA3 directly cooperate with TALE TFs and selectively target different subsets of a broad TALE chromatin platform. Binding of HOX and tissue-specific TFs convert low affinity TALE binding into high confidence, tissue-specific binding events, which bear the mark of active enhancers. We propose that HOX paralogs, alone and in combination with tissue-specific TFs, generate tissue-specific transcriptional outputs by modulating the activity of TALE TFs at selected enhancers., Competing Interests: The authors declare no competing interests.

Objectives: This article aims to: (1) describe the 'Return to Open Pharmacy Operations' in Puerto Rico following the hurricanes Irma and Maria in the 2017 hurricane season, and (2) compare the recovery rate (Return to Open Pharmacy Operations) during the 2017 hurricane season between the US Commonwealth of Puerto Rico and the state of Florida., Methods: We performed a cross-sectional study of pharmacy operations in Puerto Rico utilizing Rx Open data for pharmacies in Puerto Rico during the 2017 hurricane season. To compare open rates of pharmacy operations over time in different contexts, we also analyzed Rx Open data for the state of Florida for Hurricane Irma., Results: Only 11.1% of pharmacies remained open in Puerto Rico 3 days after Hurricane Maria made landfall, and Puerto Rico pharmacy operations recovered slowly, at an average daily rate of 3.9% before reaching pre-landfall baseline operations. Puerto Rico pharmacy operations after Hurricane Maria recovered 10 times slower on average, compared to pharmacy operations in Florida after Hurricane Irma which reached baseline operations less than 1 week following Hurricane Irma's landfall., Conclusion: Our results demonstrate the unique severity of Hurricane Maria's impacts on Puerto Rico's health system.

Niemann-Pick type C1 (NPC1) is a large multidomain transmembrane protein essential for transporting cholesterol (CLR) from late endosomes and lysosomes to the endoplasmic reticulum and other cellular compartments. The lumen-facing N-terminal domain (NTD), involved in direct binding of CLR, is expected to have an optimum activity at acidic pH = 4.5. Here, we show that acidic pH is vital for the functionality of NPC1(NTD) and should be taken into account when studying the protein activity. We applied evolutionary, structural, and physicochemical analyses to decipher the consequences of a change in pH from acidic (pH = 4.5) to neutral (pH = 7.2) on the structural integrity of the NTD and its ability to bind CLR. We revealed that the change in pH from 4.5 to 7.2 increases the potential energy of the protein in both apo- and holo-states making the system less energetically favorable. At neutral pH, the flexibility of the protein in the apo-state is decreased caused by the alteration of specific interactions, which in turn might have a high impact on ligand recognition and binding. In contrast, neutral pH significantly exaggerates the flexibility of the protein with bound CLR that causes a partial exposure of the ligand to the water phase and its mislocation inside the ligand-binding pocket, which might obstruct CLR translocation through the membrane., Competing Interests: The authors declare no competing financial interest., (© 2020 American Chemical Society.)

Background: Chronic myelomonocytic leukaemia (CMML) is a clinically heterogeneous stem cell malignancy with overlapping features of myelodysplasia and myeloproliferation. Over 90% of patients carry mutations in epigenetic and/or splicing genes, typically detectable in the Lin - CD34 + CD38 - immunophenotypic stem cell compartment in which the leukaemia-initiating cells reside. Transcriptional dysregulation at the stem cell level is likely fundamental to disease onset and progression., Methods: We performed single-cell RNA sequencing on 6826 Lin - CD34 + CD38 - stem cells from CMML patients and healthy controls using the droplet-based, ultra-high-throughput 10x platform., Findings: We found substantial inter- and intra-patient heterogeneity, with CMML stem cells displaying distinctive transcriptional programs. Compared with normal controls, CMML stem cells exhibited transcriptomes characterized by increased expression of myeloid-lineage and cell cycle genes, and lower expression of genes selectively expressed by normal haematopoietic stem cells. Neutrophil-primed progenitor genes and a MYC transcription factor regulome were prominent in stem cells from CMML-1 patients, whereas CMML-2 stem cells exhibited strong expression of interferon-regulatory factor regulomes, including those associated with IRF1, IRF7 and IRF8. CMML-1 and CMML-2 stem cells (stages distinguished by proportion of downstream blasts and promonocytes) differed substantially in both transcriptome and pseudotime, indicating fundamentally different biology underpinning these disease states. Gene expression and pathway analyses highlighted potentially tractable therapeutic vulnerabilities for downstream investigation. Importantly, CMML patients harboured variably-sized subpopulations of transcriptionally normal stem cells, indicating a potential reservoir to restore functional haematopoiesis., Interpretation: Our findings provide novel insights into the CMML stem cell compartment, revealing an unexpected degree of heterogeneity and demonstrating that CMML stem cell transcriptomes anticipate disease morphology, and therefore outcome., Funding: Project funding was supported by Oglesby Charitable Trust, Cancer Research UK, Blood Cancer UK, and UK Medical Research Council., Competing Interests: Declaration of Compeitng Interest Authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Pseudomonas aeruginosa is an opportunistic human pathogen capable of causing a wide range of potentially life-threatening infections. With multidrug-resistant P. aeruginosa infections on the rise, the need for a rationally-designed vaccine against this pathogen is critical. A number of vaccine platforms have shown promising results in pre-clinical studies, but no vaccine has successfully advanced to licensure. Growing evidence suggests that an effective P. aeruginosa vaccine may require Th17-type CD4 + T cells to prevent infection. In this review, we summarize recent pre-clinical studies of P. aeruginosa vaccines, specifically focusing on those that induce Th17-type cellular immunity. We also highlight the importance of adjuvant selection and immunization route in vaccine design in order to target vaccine-induced immunity to infected tissues. Advances in cellular immunology and adjuvant biology may ultimately influence better P. aeruginosa vaccine platforms that can protect targeted human populations.

Burkholderia cepacia complex (Bcc) lung infections in cystic fibrosis (CF) patients are often associated with a steady decline in lung function and death. The formation of biofilms and inherent multidrug resistance are virulence factors associated with Bcc infection and contribute to increased risk of mortality in CF patients. New therapeutic strategies targeting bacterial biofilms are anticipated to enhance antibiotic penetration and facilitate resolution of infection. Poly (acetyl, arginyl) glucosamine (PAAG) is a cationic glycopolymer therapeutic being developed to directly target biofilm integrity. In this study, 13 isolates from 7 species were examined, including Burkholderia multivorans , Burkholderia cenocepacia , Burkholderia gladioli , Burkholderia dolosa , Burkholderia vietnamiensis , and B. cepacia These isolates were selected for their resistance to standard clinical antibiotics and their ability to form biofilms in vitro Biofilm biomass was quantitated using static tissue culture plate (TCP) biofilm methods and a minimum biofilm eradication concentration (MBEC) assay. Confocal laser scanning microscopy (CLSM) visualized biofilm removal by PAAG during treatment. Both TCP and MBEC methods demonstrated a significant dose-dependent relationship with regard to biofilm removal by 50 to 200 μg/ml PAAG following a 1-h treatment ( P  < 0.01). A significant reduction in biofilm thickness was observed following a 10-min treatment of Bcc biofilms with PAAG compared to that with vehicle control ( P  < 0.001) in TCP, MBEC, and CLSM analyses. PAAG also rapidly permeabilizes bacteria within the first 10 min of treatment. Glycopolymers, such as PAAG, are a new class of large-molecule therapeutics that support the treatment of recalcitrant Bcc biofilm., (Copyright © 2019 Narayanaswamy et al.)

Cystic fibrosis (CF) is characterized by increased mucus viscosity and delayed mucociliary clearance that contributes to progressive decline of lung function. Mucus in the respiratory and GI tract is excessively adhesive in the presence of airway dehydration and excess extracellular Ca2+ upon mucin release, promoting hyperviscous, densely packed mucins characteristic of CF. Therapies that target mucins directly through ionic interactions remain unexploited. Here we show that poly (acetyl, arginyl) glucosamine (PAAG), a polycationic biopolymer suitable for human use, interacts directly with mucins in a Ca2+-sensitive manner to reduce CF mucus viscoelasticity and improve its transport. Notably, PAAG induced a linear structure of purified MUC5B and altered its sedimentation profile and viscosity, indicative of proper mucin expansion. In vivo, PAAG nebulization improved mucociliary transport in CF rats with delayed mucus clearance, and cleared mucus plugging in CF ferrets. This study demonstrates the potential use of a synthetic glycopolymer PAAG as a molecular agent that could benefit patients with a broad array of mucus diseases.

Alcohols, aldehydes, ketones, amines, and sulfur compounds are essential aroma compounds related to fish flavor and spoilage. Gas chromatography-mass spectrometry (GC-MS) is an instrument that is widely used to identify and quantify volatile and semi-volatile compounds in fish products. In this research, a simple and accurate GC-MS method was developed to determine the aroma profile of mahi-mahi and tuna for chemical indicators of spoilage. In the developed GC-MS method, trichloroacetic acid (TCA) solution was used to extract analytes from homogenized fish samples. The purge and trap system was used for sample introduction, and the GC-MS with an RTX-Volatile Amine column was able to separate compounds without a derivatization procedure. The created purge and trap gas chromatography-mass spectrometry (PT-GC-MS) method could identify and quantify twenty aroma compounds in mahi-mahi (Coryphaena hippurus) and 16 volatile compounds in yellowfin tuna (Thunnus albacares) associated with fish spoilage. The amines (dimethylamine, trimethylamine, isobutylamine, 3-methylbutylamine, and 2-methylbutanamine), alcohols (2-ethylhexanol, 1-penten-3-ol and isoamyl alcohol, ethanol), aldehydes (2-methylbutanal, 3-methylbutanal, benzaldehyde), ketones (acetone, 2,3-butanedione, 2-butanone, acetoin), and dimethyl disulfide strongly statistically correlated with poorer quality tuna and mahi-mahi and were considered as the key spoilage indicators. PRACTICAL APPLICATION: A simplified and rapid purge and trap gas chromatography-mass spectrometry (PT-GC-MS) method developed in this research was able to identify and quantify important spoilage compounds in mahi-mahi and yellowfin tuna. This method is an efficient analytical method for determining volatile profiles of fish samples for industry analytical labs or the government. The identified analytical quality markers can be used to monitor the spoilage level of tuna and mahi-mahi., (© 2019 Institute of Food Technologists®.)

Respiratory infections are a leading cause of morbidity and mortality globally. This is partially due to a lack of effective vaccines and a clear understanding of how vaccination route and formulation influence protective immunity in mucosal tissues such as the lung. Pseudomonas aeruginosa is an opportunistic pathogen capable of causing acute pulmonary infections and is a leading cause of hospital-acquired and ventilator-associated pneumonia. With multidrug-resistant P. aeruginosa infections on the rise, the need for a vaccine against this pathogen is critical. Growing evidence suggests that a successful P. aeruginosa vaccine may require mucosal antibody and Th1- and Th17-type CD4 + T cells to prevent pulmonary infection. Intradermal immunization with adjuvants, such as the bacterial ADP-Ribosylating Enterotoxin Adjuvant (BARE) double mutant of E. coli heat-labile toxin (dmLT), can direct protective immune responses to mucosal tissues, including the lungs. We reasoned that intradermal immunization with P. aeruginosa outer membrane proteins (OMPs) adjuvanted with dmLT could drive neutralizing antibodies and migration of CD4 + T cells to the lungs and protect against P. aeruginosa pneumonia in a murine model. Here we show that mice immunized with OMPs and dmLT had significantly more antigen-specific IgG and Th1- and Th17-type CD4 + memory T cells in the pulmonary environment compared to control groups of mice. Furthermore, OMPs and dmLT immunized mice were significantly protected against an otherwise lethal lung infection. Protection was associated with early IFN-γ and IL-17 production in the lungs of immunized mice. These results indicate that intradermal immunization with dmLT can drive protective immunity to the lung mucosa and may be a viable vaccination strategy for a multitude of respiratory pathogens., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

ATAC-seq is a recently developed method to identify the areas of open chromatin in a cell. These regions usually correspond to active regulatory elements and their location profile is unique to a given cell type. When done at single-cell resolution, ATAC-seq provides an insight into the cell-to-cell variability that emerges from otherwise identical DNA sequences by identifying the variability in the genomic location of open chromatin sites in each of the cells. This paper presents Scasat (single-cell ATAC-seq analysis tool), a complete pipeline to process scATAC-seq data with simple steps. Scasat treats the data as binary and applies statistical methods that are especially suitable for binary data. The pipeline is developed in a Jupyter notebook environment that holds the executable code along with the necessary description and results. It is robust, flexible, interactive and easy to extend. Within Scasat we developed a novel differential accessibility analysis method based on information gain to identify the peaks that are unique to a cell. The results from Scasat showed that open chromatin locations corresponding to potential regulatory elements can account for cellular heterogeneity and can identify regulatory regions that separates cells from a complex population.

Background: Post-operative infection (POI) is a serious complication in all surgical disciplines and can derail a patient's treatment and recovery course. In this analysis, we examine national trends, risk factors, and costs associated with POI., Methods: Using the Nationwide Inpatient Sample (NIS) for the years available for data analysis at the time of this study (2003-2010), we performed a cross-sectional study of adult (≥18 years) inpatients with POI and designated Clinical Classification Software (CCS) procedural class codes for the operations performed. A comparison group was selected randomly from patients with the same CCS codes who underwent the same procedures but did not experience POI. As the NIS represents 20% of U.S. hospital admissions, excess cost and stay were calculated on the basis of the average difference between cost and duration of stay for POI cases and the cost and duration of stay for the comparison group, then extrapolated to estimate the national burden for the remaining 80% of stays nationwide., Results: Sample admissions included 139,652 cases of POI and 941,670 comparison subjects. The POIs were most common in procedures involving the digestive tract (46.5%), cardiovascular system (16.3%), or musculoskeletal system (11.2%). Older age, male gender, high Charlson Comorbidity Index Score (CCIS), and teaching, urban, or large hospitals were independent risk factors for POI in the multivariable model (p < 0.05). A POI was associated with a higher risk of death (odds ratio 2.93; 95% confidence interval 2.82-3.04, p < 0.001). Nationally, we estimate that POI resulted in an annual average of 1.04 million days of excess hospital stay and $2.72 billion excess cost., Conclusions: Identification of independent risk factors suggests areas for quality improvement initiatives. Post-operative infection carries substantial clinical and financial burdens in the United States, and further analysis of the associated costs is needed to identify areas for intervention to reduce this burden.

Cerebral malaria (CM) is a serious neurological complication caused by Plasmodium falciparum infection. Currently, the only treatment for CM is the provision of antimalarial drugs; however, such treatment by itself often fails to prevent death or development of neurological sequelae. To identify potential improved treatments for CM, we performed a nonbiased whole-brain transcriptomic time-course analysis of antimalarial drug chemotherapy of murine experimental CM (ECM). Bioinformatics analyses revealed IL33 as a critical regulator of neuroinflammation and cerebral pathology that is down-regulated in the brain during fatal ECM and in the acute period following treatment of ECM. Consistent with this, administration of IL33 alongside antimalarial drugs significantly improved the treatment success of established ECM. Mechanistically, IL33 treatment reduced inflammasome activation and IL1β production in microglia and intracerebral monocytes in the acute recovery period following treatment of ECM. Moreover, treatment with the NLRP3-inflammasome inhibitor MCC950 alongside antimalarial drugs phenocopied the protective effect of IL33 therapy in improving the recovery from established ECM. We further showed that IL1β release from macrophages was stimulated by hemozoin and antimalarial drugs and that this was inhibited by MCC950. Our results therefore demonstrate that manipulation of the IL33-NLRP3 axis may be an effective therapy to suppress neuroinflammation and improve the efficacy of antimalarial drug treatment of CM., Competing Interests: The authors declare no conflict of interest.

Using consensual qualitative research (CQR), we analyzed 13 interviews of experienced psychotherapists about general intentions for therapist self-disclosure (TSD), experiences with successful TSDs, experiences with unsuccessful TSDs, and instances of unmanifested urges to disclose. For TSD generally (i.e., not about a specific instance), typical intentions were to facilitate exploration and build and maintain the therapeutic relationship. Therapists typically reported becoming more comfortable using TSD over time. In successful TSDs, the typical content was accurate, relevant similarities between therapist and client; typical consequences were positive. In unsuccessful TSDs, the typical antecedent was countertransference reactions; the typical intention was to provide support; typical content involved therapists mistakenly perceiving similarities with clients; and the general consequences were negative. In instances when therapists repressed the urge to disclose, the typical antecedent was countertransference and the content typically seemed relevant to the client's issues. We conclude that effective use of TSD requires general attunement to the client's dynamics, attunement to the client's readiness in the moment, ability to manage countertransference, and ability to use a specific TSD appropriately. Implications for practice, training, and research are discussed. (PsycINFO Database Record, ((c) 2018 APA, all rights reserved).)

The incidence of multidrug-resistant (MDR) organisms, including methicillin-resistant Staphylococcus aureus (MRSA), is a serious threat to public health. Progress in developing new therapeutics is being outpaced by antibiotic resistance development, and alternative agents that rapidly permeabilize bacteria hold tremendous potential for treating MDR infections. A new class of glycopolymers includes polycationic poly-N (acetyl, arginyl) glucosamine (PAAG) is under development as an alternative to traditional antibiotic strategies to treat MRSA infections. This study demonstrates the antibacterial activity of PAAG against clinical isolates of methicillin and mupirocin-resistant Staphylococcus aureus. Multidrug-resistant S. aureus was rapidly killed by PAAG, which completely eradicated 88% (15/17) of all tested strains (6-log reduction in CFU) in ≤ 12-hours at doses that are non-toxic to mammalian cells. PAAG also sensitized all the clinical MRSA strains (17/17) to oxacillin as demonstrated by the observed reduction in the oxacillin MIC to below the antibiotic resistance breakpoint. The effect of PAAG and standard antibiotics including vancomycin, oxacillin, mupirocin and bacitracin on MRSA permeability was studied by measuring propidium iodide (PI) uptake by bacterial cells. Antimicrobial resistance studies showed that S. aureus developed resistance to PAAG at a rate slower than to mupirocin but similar to bacitracin. PAAG was observed to resensitize drug-resistant S. aureus strains sampled from passage 13 and 20 of the multi-passage resistance study, reducing MICs of mupirocin and bacitracin below their clinical sensitivity breakpoints. This class of bacterial permeabilizing glycopolymers may provide a new tool in the battle against multidrug-resistant bacteria.

Burkholderia mallei is a Gram-negative, non-motile, facultative intracellular bacillus and the causative agent of glanders, a highly contagious zoonotic disease. B. mallei is naturally resistant to multiple antibiotics and there is concern for its potential use as a bioweapon, making the development of a vaccine against B. mallei of critical importance. We have previously demonstrated that immunization with multivalent outer membrane vesicles (OMV) derived from B. pseudomallei provide significant protection against pneumonic melioidosis. Given that many virulence determinants are highly conserved between the two species, we sought to determine if the B. pseudomallei OMV vaccine could cross-protect against B. mallei . We immunized C57Bl/6 mice and rhesus macaques with B. pseudomallei OMVs and subsequently challenged animals with aerosolized B. mallei . Immunization with B. pseudomallei OMVs significantly protected mice against B. mallei and the protection observed was comparable to that achieved with a live attenuated vaccine. OMV immunization induced the production of B.mallei- specific serum IgG and a mixed Th1/Th17 CD4 and CD8 T cell response in mice. Additionally, immunization of rhesus macaques with B. pseudomallei OMVs provided protection against glanders and induced B.mallei -specific serum IgG in non-human primates. These results demonstrate the ability of the multivalent OMV vaccine platform to elicit cross-protection against closely-related intracellular pathogens and to induce robust humoral and cellular immune responses against shared protective antigens., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

After the publication of this work [1] it was noticed that on page 3 of the article beginning in the paragraph-'Hydrazide 14 is used as a key producer…' the compound-"Not ethyl-N'-5-amino-1,3-diphenyl-1H-thieno[3,2-c]pyrazole-6-carbonylformohydrazonate" should be deleted. Also on page 10 of the PDF beginning in paragraph-'Synthesis of 5-amino-…' the compound-"Not ethyl-N'-5-amino-1,3-diphenyl-1H-thieno[3,2-c]pyrazole-6-carbonylformohydrazonate" should be deleted. We apologise for these errors.

Background: Some novel substituted pyrazolone, pyrazolo[3,4-c]pyridazine, pyrazolo[3,4-d]pyrimidine, pyrazolo[3,4-d]thiazolo[3,2-a]pyrimidinone, thieno[3,2-c]pyrazole and pyrazolo[3',4':4,5]thieno[2,3-d]pyrimidine derivatives have been reported to possess various pharmacological activities like antimicrobial, antitumor and anti-inflammatory., Results: A novel series of azoles and azines were designed and prepared via reaction of 1,3-diphenyl-1H-pyrazol-5(4H)-one with some electrophilic and nucleophilic reagents. The structures of target compounds were confirmed by elemental analyses and spectral data., Conclusions: The antimicrobial activity of the target synthesized compounds were tested against various microorganisms such as Escherichia coli; Bacillus megaterium; Bacillus subtilis (Bacterial species), Fusarium proliferatum; Trichoderma harzianum; Aspergillus niger (fungal species) by the disc diffusion method. In general, the novel synthesized compounds showed a good antimicrobial activity against the previously mentioned microorganisms.

Burkholderia cepacia complex (Bcc) infection, associated with cystic fibrosis (CF) is intrinsically multidrug resistant to antibiotic treatment making eradication from the CF lung virtually impossible. Infection with Bcc leads to a rapid decline in lung function and is often a contraindication for lung transplant, significantly influencing morbidity and mortality associated with CF disease. Standard treatment frequently involves antibiotic combination therapy. However, no formal strategy has been adopted in clinical practice to guide successful eradication. A new class of direct-acting, large molecule polycationic glycopolymers, derivatives of a natural polysaccharide poly-N-acetyl-glucosamine (PAAG), are in development as an alternative to traditional antibiotic strategies. During treatment, PAAG rapidly targets the anionic structural composition of bacterial outer membranes. PAAG was observed to permeabilize bacterial membranes upon contact to facilitate potentiation of antibiotic activity. Three-dimensional checkerboard synergy analyses were used to test the susceptibility of eight Bcc strains (seven CF clinical isolates) to antibiotic combinations with PAAG or ceftazidime. Potentiation of tobramycin and meropenem activity was observed in combination with 8-128 μg/mL PAAG. Treatment with PAAG reduced the minimum inhibitory concentration (MIC) of tobramycin and meropenem below their clinical sensitivity breakpoints (≤4 μg/mL), demonstrating the ability of PAAG to sensitize antibiotic resistant Bcc clinical isolates. Fractional inhibitory concentration (FIC) calculations showed PAAG was able to significantly potentiate antibacterial synergy with these antibiotics toward all Bcc species tested. These preliminary studies suggest PAAG facilitates a broad synergistic activity that may result in more positive therapeutic outcomes and supports further development of safe, polycationic glycopolymers for inhaled combination antibiotic therapy, particularly for CF-associated Bcc infections.